Bacteria play critical roles in biogeochemical cycles, such as the fixation of nitrogen. Although nitrogen makes up approximately 80% of the Earth's atmosphere, its bioavailability remains a major limitation, in particular to plant growth. This is due to the inability of plants to assimilate the diatomic nitrogen that occurs naturally in the atmosphere. Among plants, legumes uniquely host bacteria in nodules formed following invasion of their root cortical cells. These bacteria are typically from the Rhizobiales order of the Alphaproteobacteria, although others are from the Betaproteobacteria class, collectively these are referred to as rhizobia or rhizobacteria. Inside these nodules, the rhizobacteria develop into endosymbiont bacteroids, fixing nitrogen in exchange for carbon from their plant hosts. This agriculturally important collaboration is thought to be the main biological route for nitrogen fixation. For example, the relationship between soybean Glycine max and the associated nodulating rhizobacteria Bradyrhizobium japonicum is essential for the critical nitrogen-fixating properties that make this crop plant so agriculturally important. Of particular relevance here, a number of the rhizobacteria have been shown to produce plant growth hormones, such as the gibberellins, which are thought to further promote growth of the host plant.
Both legume and rhizobacterial species exhibit a surprising amount of specificity with respect to symbiotic partners. Only rarely can a given rhizobacterial species nodulate more than a few closely related plants. This specificity is due to bacterial and plant factors. The host plant secretes flavonoid inducers that elicit rhizobacterial production of lipochitooligosaccharide Nod factors, which are recognized by the host plant, with subsequent steps in the nodulation process being dependent on recognition of bacterial cell surface chemistry and effector proteins as well. However, the host plant also applies the usual defense mechanisms—e.g., microbe associated molecular pattern-triggered immunity and R-gene recognition of bacterial effectors—to restrict nodulation by unwanted strains. This complex signal exchange process exerts extreme evolutionary pressure on the rhizobacteria, which can be inferred, in part, from the presence of large plasmids or genomic islands with distinct G+C contents relative to the G+C content in the rest of the genome. These large plasmids or genomic islands contain the large set of genes required for nodulation, including nitrogen fixation. This presumably reflects the ability of horizontal transfer to spread these distinct genetic elements, enabling nodulation by the recipient rhizobacteria. Intriguingly, the prevalence of insertion sequences and phage integration is thought to promote rearrangement within these symbiotic nodules.
The nitrogen-fixing capacity imparted to soybean via nodulation of their roots by B. japonicum offers a clear advantage to the plant. Indeed, it is this nitrogen fixation that is largely responsible for the benefits of including soybean as a rotational crop. Such nodulation occurs as the product of highly specific partnerships formed between individual leguminous plant species and a particular species of rhizobacteria. This involves providing the rhizobacterial partner not only access to the interior of the host plant root, but also a hospitable environment, including feeding with photosynthetically fixed carbon. Intriguingly, presumably due to the dangers of establishing such an intimate relationship with a microbe, most legumes actually force their nodulating rhizobacterial partner to undergo a transition from the free-living form found in the soil to a terminally differentiated bacteriod form that essentially becomes a nitrogen-fixing organelle, and cannot go back to living in the soil. However, this is not true in soybean and other determinate nodule forming legumes, where the symbiotic microbe (e.g., B. japonicum for soybean) retains its usual form, and can go back to living in the soil.
The gibberellins are a large group of complex diterpenoid natural products, among which several have potent biological activity in plants, where they serve as hormones. Intriguingly, these phytohormones are made not only by the plants in which they serve to regulate growth and development, but by certain plant associated fungal and bacterial microbes as well. While gibberellin phytohormone biosynthesis has been largely elucidated for higher plants and fungi, which seem to have independently evolved/assembled the corresponding metabolic pathway, the basis for such biosynthesis in bacteria remains enigmatic. Even in higher plants, the origins of gibberellin metabolism remains obscure. Further, there have been recent discoveries demonstrating the existence of novel gibberellin metabolism (particularly catabolism) in higher plants, which have critical yet unexplored implications for flux in and the regulation of gibberellin metabolism.
Two terpene synthases have been characterized from a strain of B. japonicum (USDA110). These proved to be diterpene cyclases capable of successively converting the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) into ent-copalyl diphosphate (ent-CPP) and, hence, to ent-kaurene, a precursor to the gibberellin phytohormones (FIG. 1). The relevant genes, blr2149 and blr2150, encode an ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS), respectively. Notably, these two genes fall into a more extensive operon that was originally defined by Tully et al. (Appl. Environ. Microbiol. 1993, 59(12):4136) and suggested to be present in all rhizobia.
Gibberellins have played an important role in agriculture, as it was alterations in such phytohormone metabolism that led to high yielding semi-dwarf varieties of rice and wheat, which were a critical component of the “Green Revolution.” The biosynthesis of gibberellin phytohormones by plant growth promoting bacteria that are commonly applied to legume crop plants offers additional significance. Further, the absolute requirement for gibberellin production in higher plants has provided a genetic reservoir of biosynthetic genes, duplication of which has led to a vast super-family (−7,000 known) of related diterpenoid natural products, exhibiting various biological activities and physiological roles (e.g., as defensive antibiotics). The production of gibberellins by B. japonicum has long been thought to promote plant growth as part of a symbiotic relationship. Although the genetic locus encoding the cellular machinery responsible for gibberellin production has previously been described, for example by Tully and Keister (Appl. Environ. Microbiol. 1993, 59(12):4136), the true role of bacterial gibberellin production has remained unclear.
Therefore, it is a primary object, feature, or advantage of the present invention to improve upon the state of the art.
It is a further object, feature, or advantage of the present invention to provide methods of altering the physiology of a plant, including enhancing pathogen resistance.
It is a further objective, feature or advantage of the present invention to provide methods for modifying rhizobacteria to eliminate or decrease bacterial gibberellin production.
It is a further objective, feature or advantage of the present invention to provide modified rhizobacteria that produce little or no gibberellin.
It is a further objective, feature or advantage of the present invention to provide methods of enhancing pathogen resistance in plants by reducing or eliminating gibberellin production by rhizobacteria in the nodules of the plants.
It is a further objective, feature or advantage of the present invention to provide rhizobacteria that have been modified to promote enhanced pathogen resistance in plants while still providing all the nitrogen-fixing benefits of nodulation.
It is a further objective, feature or advantage of the present invention to provide compositions comprising a nodulating plant and a modified rhizobacteria wherein the plant has altered physiology as a result of the modified rhizobacteria.